Above pile-up fluorescence microscopy with a 32 Mc/s single-channel time-resolved SPAD system.

One of the major drawbacks of time-correlated single-photon counting (TCSPC) is generally represented by pile-up distortion, which strongly bounds the maximum acquisition speed to a few percent of the laser excitation rate. Based on a previous theoretical analysis, recently we presented the first, to the best of our knowledge, low-distortion and high-speed TCSPC system capable of overcoming the pile-up limitation by perfectly matching the single-photon avalanche diode (SPAD) dead time to the laser period. In this work, we validate the proposed system in a standard fluorescence measurement by comparing experimental data with the reference theoretical framework. As a result, a count rate of 32 Mc/s was achieved with a single-channel system still observing a negligible lifetime distortion.

Multispectral compressive fluorescence lifetime imaging microscopy with a SPAD array detector.

Multispectral/hyperspectral fluorescence lifetime imaging microscopy (λFLIM) is a promising tool for studying functional and structural biological processes. The rich information content provided by a multidimensional dataset is often in contrast with the acquisition speed. In this work, we develop and experimentally demonstrate a wide-field λFLIM setup, based on a novel time-resolved 18×1 single-photon avalanche diode array detector working in a single-pixel camera scheme, which parallelizes the spectral detection, reducing measurement time. The proposed system, which implements a single-pixel camera with a compressive sensing scheme, represents an optimal microscopy framework towards the design of λFLIM setups.

Custom-Technology Single-Photon Avalanche Diode Linear Detector Array for Underwater Depth Imaging.

We present an optical depth imaging system suitable for highly scattering underwater environments. The system used the time-correlated single-photon counting (TCSPC) technique and the time-of-flight approach to obtain depth profiles. The single-photon detection was provided by a linear array of single-photon avalanche diode (SPAD) detectors fabricated in a customized silicon fabrication technology for optimized efficiency, dark count rate, and jitter performance. The bi-static transceiver comprised a pulsed laser diode source with central wavelength 670 nm, a linear array of 16 × 1 Si-SPAD detectors, with a dedicated TCSPC acquisition module. Cylindrical lenses were used to collect the light scattered by the target and image it onto the sensor. These laboratory-based experiments demonstrated single-photon depth imaging at a range of 1.65 m in highly scattering conditions, equivalent up to 8.3 attenuation lengths between the system and the target, using average optical powers of up to 15 mW. The depth and spatial resolution of this sensor were investigated in different scattering conditions.

Red-Enhanced Photon Detection Module Featuring a 32 × 1 Single-Photon Avalanche Diode Array.

In this letter, the development and the experimental characterization of a new photon detection module, based on a 32×1 red-enhanced single-photon avalanche diode (RE-SPAD) array, are presented. A custom-developed technology has been exploited to design a detector having large-area pixels (50-µm diameter) with optimized performance. With an excess bias voltage Voυ = 15 V, a photon detection efficiency as high as 57% at 600 nm (33% at 800 nm) is achieved, along with dark count rate in the kHz range and optical crosstalk probability as low as 0.29%. The remarkable detection efficiency of the RE-SPAD array makes the module particularly suitable for all applications where high detection efficiency in the red/near-infrared range is mandatory. As an example, the performance of the array module is demonstrated to match the demanding requirements of multispot single-molecule fluorescence spectroscopy.

48-spot single-molecule FRET setup with periodic acceptor excitation.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Gigacount/second Photon Detection Module Based on an 8 × 8 Single-Photon Avalanche Diode Array.

In this letter we present a compact photon detection module, based on an 8×8 array of single-photon avalanche diodes (SPADs). The use of a dedicated silicon technology for the fabrication of the sensors allows us to combine large active areas (50-µm diameter), high photon detection efficiency (49% at 550-nm wavelength) and low dark count rate. Thanks to a fully parallel architecture, the module provides voltage pulses synchronous to each photon detection for a maximum global count rate exceeding 1 Gcps. These properties makes the system suitable for operation in two different free-running modes. The first, suitable to acquire faint signals, allows multi-spot acquisitions and can be used to considerably reduce the measurement time in applications like single-molecule analysis. With the second it is possible to use all the pixels in a combined mode, to extend and move the dynamic range of the module to very high count rates and to attain number resolving capabilities.

4 ns dead time with a fully integrated active quenching circuit driving a custom single photon avalanche diode.

At the present time, Single Photon Avalanche Diodes (SPADs) are the enabling devices in many applications, ranging from medical imaging to laser ranging and from remote sensing to quantum key distribution. Even though they belong to different scientific domains, these applications share the need for a detector capable of attaining high count rates possibly without trading it off with other key detector's features, such as afterpulsing probability, photon detection efficiency, and dark counts. In this work, we present the characterization of a fast integrated active quenching circuit capable of driving high-performance external custom-technology SPADs for single photon detection in the visible wavelength range. Combining the prompt intervention of the electronic circuitry and the performance of a custom-technology SPAD, we attained count rates up to 250 MCps while keeping the afterpulsing probability within 2%.

Optical crosstalk in single photon avalanche diode arrays: a new complete model.

One of the main issues of Single Photon Avalanche Diode arrays is optical crosstalk. Since its intensity increases with reducing the distance between devices, this phenomenon limits the density of integration within arrays. In the past optical crosstalk was ascribed essentially to the light propagating from one detector to another through direct optical paths. Accordingly, reflecting trenches between devices were proposed to prevent it, but they proved to be not completely effective. In this paper we will present experimental evidence that a significant contribution to optical crosstalk comes from light reflected internally off the bottom of the chip, thus being impossible to eliminate it completely by means of trenches. We will also propose an optical model to predict the dependence of crosstalk on the distance between devices.

Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10-3) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Operation of silicon single photon avalanche diodes at cryogenic temperature.

This article reports a complete characterization of single photon avalanche diodes (SPADs) at temperatures down to 120 K. We show that deep cooling of the device by means of a compact liquid-nitrogen Dewar brings several advantages, such as extremely low dark counting rates (down to 1 counts/s), better time resolution, and higher quantum efficiency in the visible range. By using a special current pick-off circuit, we achieved a time resolution of 20 ps full width at half maximum at 120 K for a 50 mum diameter SPAD. Afterpulsing effects are avoided by using a sufficiently long hold-off time (microseconds).

Development and characterization of an 8×8 SPAD-array module for gigacount per second applications.

In recent years the development of Single-Photon Avalanche Diodes (SPADs) had a big impact on single-photon counting applications requiring high-performance detectors in terms of Dark Count Rate (DCR), Photon Detection Efficiency (PDE), afterpulsing probability, etc. Among these, it is possible to find applications in single-molecule fluorescence spectroscopy that suffer from long-time measurements. In these cases SPAD arrays can be a solution in order to shorten the measurement time, thanks to the high grade of parallelism they can provide. Moreover, applications in other fields (e.g. astronomy) demand for large-area single-photon detectors, able also to handle very high count rates. For these reasons we developed a new single-photon detection module, featuring an 8 × 8 SPAD array. Thanks to a dedicated silicon technology, the performance of the detector have been finely optimized, reaching a 49% detection efficiency at 550 nm, as well as low dark counts (2 kcount/s maximum all over the array). This module can be used in two different modes: the first is a multi-spot configuration, allowing the acquisition of 64 optical signals at the same time and considerably reducing the time needed for a measurement. The second operation mode instead exploits all the pixels in a combined mode, allowing the detection of a 64-times higher maximum photon rate (up to 2 Gcount/s). In addition, this configuration provides also an extended dynamic range and allows to attain photon number resolving capabilities. Dark counts, detection efficiency, linearity, afterpulsing and crosstalk probability have been characterized at different operating conditions.